Enzymes in the a-D-phosphohexomutase superfamily play critical roles in carbohydrate metabolism and other biosynthetic pathways. The biological importance of these proteins is demonstrated by their distribution in nature: at least one family member appears to be found in all organisms from bacteria to humans. In this proposal, we will characterize novel aspects of one protein in this enzyme family, phosphomannomutase/phosphoglucomutase (PMM/PGM) from P. aeruginosa. This bacterium is an opportunistic human pathogen that often infects cystic fibrosis patients, burn victims, and cancer patients. PMM/PGM participates in the biosynthesis of multiple bacterial exoproducts, including lipopolysaccharide, alginate and rhamnolipid, which are associated with biofilm formation and increased virulence in P. aeruginosa infections. Because of its key role in the biosynthetic pathways of virulence factors, PMM/PGM is a target for inhibitor design. Previous work has shown that conformational change of this enzyme is required at several key points in its multi-step reaction, which entails two consecutive phosphoryl transfer reactions with an intervening reorientation of the reaction intermediate. Recently, other laboratories have shown evidence for a new paradigm of enzyme dynamics in catalysis: intrinsic fluctuations appear to couple to catalysis. We will test this paradigm in the processive enzyme PMM/PGM. We will use NMR relaxation dispersion to characterize the dynamics of substrate-free (apo) PMM/PGM that occur on a catalytically relevant timescale. In addition, we will initiate investigation of its dynamics during its reversible catalytic reaction. We seek to determine the relationship between protein conformational change, intrinsic fluctuations occurring within milliseconds, and function for this member of the a-D- phosphohexomutase enzyme superfamily. This will aid the long-term goal of developing novel inhibitors that can act at different points in its multi-step reaction. The work in this proposal will initiate characterization of the dynamics of a key enzyme (PMM/PGM) from the bacterial pathogen Pseudomonas aeruginosa. P. aeruginosa infections are the primary cause of mortality for cystic fibrosis patients, and this organism is also a leading cause of hospital-acquired infections. Dynamics studies of PMM/PGM will complement existing structural and mechanistic characterization, with the goal of designing specific inhibitors with utility for the treatment of P. aeruginosa infections.